Single-Molecule Dissection of mTOR Complexes

mTOR 复合物的单分子解剖

• 批准号: 8469719
• 负责人: Jie Chen
• 金额: $ 14万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-30 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8469719
• 关键词: Aging; Biochemical; Biological Assay; Cardiovascular Diseases; Cell Proliferation; Cell physiology; Cells; Chimeric Proteins; Complex; Development; Developmental Cell Biology; Developmental Process; Disease; Dissection; FDA approved; Fluorescence; Future; Health; Heterogeneity; In Situ; Investigation; Knowledge; Laboratories; Longevity; Malignant Neoplasms; Mammals; Measures; Medicine; Metabolic Diseases; Metabolism; Methods; Mitogens; Modeling; Molecular; Nutrient; Pathway interactions; Phosphorylation; Phosphotransferases; Play; Procedures; Process; RNA Interference; Regulation; Resolution; Role; Signal Transduction; Sirolimus; Structure-Activity Relationship; Tacrolimus Binding Protein 1A; Therapeutic; Time; age related; analog; cell growth; clinical application; crosslink; design; dimer; inhibitor/antagonist; insight; mTOR protein; novel; novel strategies; protein complex; public health relevance; single molecule; stoichiometry; therapeutic development

DESCRIPTION (provided by applicant): The mammalian target of rapamycin (mTOR) signaling network has emerged as a master regulator of essential processes in cell and developmental biology, including cell growth, proliferation, cellular differentiation, and metabolism. The multiple FDA-approved clinical applications of rapamycin attest to the central importance of mTOR in medicine. Recent evidence has also revealed a key role for mTOR signaling in modulating lifespan in addition to impacting health span. Molecular dissection of the mTOR regulatory network will facilitate development of therapeutics against aging and aging-related diseases, including cardiovascular diseases, metabolic diseases, and cancer. mTOR assembles two biochemically and functionally distinct protein complexes - mTORC1 and mTORC2, which nucleate distinct pathways and yet crosstalk extensively. The current knowledge of mTOR complex assembly is largely derived from conventional biochemical characterizations, which typically require lengthy and strenuous purification procedures that may disrupt native complexes, and offer limited insights into the stoichiometry and heterogeneity of complex formation. We (the Ha laboratory) have recently developed a single-molecule pull-down (SiMPull) method that enables rapid and sensitive analysis of protein complexes at single-complex resolution, directly from whole cell lysates. Here we propose to dissect the assembly of mTORC1 and mTORC2 employing the SiMPul approach. Stoichiometry of each component in the complexes will be determined. Regulation of the complexes by upstream signals, and potential physical crosstalk between the two complexes, will also be examined. Furthermore, we will develop a single-molecule kinase assay with the SiMPull platform, and apply this novel approach to interrogating the activities of various mTOR complexes. The proposed study is expected to decipher the assembly of both mTORC1 and mTORC2 at the single-complex level and reveal structure-function relationships in the mTOR assemblies. Knowledge to be gained from this study will be invaluable in guiding future investigation of the mTOR regulatory network and in facilitating potentially novel design of therapeutic strategies.

描述（由申请人提供）：雷帕霉素（MTOR）信号网络的哺乳动物靶标已成为细胞和发育生物学基本过程的主要调节剂，包括细胞生长，增殖，细胞分化和代谢。雷帕霉素的多个FDA批准的临床应用证明了MTOR在医学上的重要性。最近的证据还揭示了MTOR信号在调节寿命中的关键作用，此外还影响了健康跨度。 MTOR调节网络的分子解剖将促进针对衰老和与衰老有关的疾病（包括心血管疾病，代谢性疾病和癌症）的疗法开发。 MTOR在功能上和功能上不同的蛋白质复合物-MTORC1和MTORC2上组装了两个，它们对不同的途径有核，但却广泛地串扰。当前对MTOR复合组装的知识在很大程度上源自常规的生化特征，这些特征通常需要延长且剧烈的纯化程序，这些过程可能会破坏天然复合物，并提供有限的见解，以实现对复杂形成的化学计量和异质性的有限见解。我们（HA实验室）最近开发了一种单分子下拉（SIMPULL）方法，该方法可以直接从全细胞裂解物中以单复合分辨率对蛋白质复合物进行快速而敏感的分析。在这里，我们建议采用SIMPUL方法的MTORC1和MTORC2的组装。将确定复合物中每个组件的化学计量法。还将检查通过上游信号对复合物的调节，以及两个复合物之间的潜在物理串扰。此外，我们将使用Simpull平台开发单分子激酶测定法，并应用这种新颖的方法来询问各种MTOR复合物的活动。 拟议的研究预计将在单复合水平上解释MTORC1和MTORC2的组装，并揭示MTOR组装中的结构 - 功能关系。从这项研究中获得的知识对于指导MTOR监管网络的未来研究以及促进潜在的治疗策略设计的新设计将是无价的。